CALCIUM-BASED EXCITABILITY IN GLIAL CELLS
, Head, Section on Cell Biology and Excitability
Lynne A. Holtzclaw, BS, Research Assistant
Sundip Patel, BS, Predoctoral Fellow
We investigate signaling between neurons and glial cells in the central and peripheral nervous systems. The intimate communication between glial cells and neurons is crucial for normal brain development and seems to play a critical role in the plastic functions of the brain. Glial cells monitor and respond to neural activity by conditioning the extracellular milieu, signaling within glial cell networks, and sending signals back to neurons. In the brain, glial cell responses to neural activity take the form of propagated Ca2+ waves that spread over long distances in response to synaptic activity. Similarly, the myelinating glial cells (oligodendrocytes and Schwann cells) receive signals from the axons they myelinate, and such signals are essential for maintaining the myelin sheath. One of our objectives is to understand different modalities of cell-cell signaling and the processes that support temporal and spatial characteristics of Ca2+ signals within and between cells. A second objective is to probe the nature of glial cell signals in response to neuronal activity and the consequences of such signals for central nervous system function.
Ca2+ signaling between axons and myelinating glia
We are investigating aspects of signaling between axons and their myelinating glia (oligodendrocytes and Schwann cells) in the peripheral nervous system (PNS) and central nervous system (CNS). Initially, we focused on characterizing the distribution of Ca2+ signaling proteins in the axoglial apparatus of the nodes of Ranvier. The myelin sheath functions as an electrical insulator that, by reducing capacitance and increasing resistance, reduces current flow across the axonal membrane in the internode, thereby facilitating saltatory conduction. Myelin is formed by a highly specialized extension of myelinating glial cell (oligodendrocytes in the CNS and Schwann cells in the PNS) membranes intimately associated with axons
Beyond the concept of saltatory conduction, several studies support the view that the axon/myelin/glial cell ensemble or “nodal complex” operates in an integrated manner during conduction of the nerve impulse. Numerous anatomical and physiological studies over the last two decades have suggested that an intricate set of signals might operate between the components of the nodal complex. Details of such signaling between axons and the myelinating glia, however, are unknown.
We are attempting to describe the distribution of proteins involved in Ca2+ signaling in the axoglial apparatus. We believed that such a description would point us to the spatial discreteness, if any, in the signaling between axons and Schwann cells. Our initial hypothesis posited that the signaling is localized to specific contact sites between the two cell types where the insulating layer of compact myelin gives way to non-compact myelin and Schwann cell membrane. We selected antibodies against proteins involved in Ca2+ signaling (e.g., IP3Rs and RyRs) and known structural proteins unique to different regions of the node and used immunocytochemistry followed by high-resolution fluorescence microscopy to investigate the proteins’ discrete localization.
We performed the analysis in an isolated, teased sciatic nerve preparation. The distribution of myelin basic protein (MBP) and S100b showed the previously described intricate architecture of the nodes of Ranvier and highlighted the intimate relationship between the axon and the myelinating glia. We focused on the arrangement of proteins in the axoglial apparatus and selected specific proteins uniquely expressed to the node (Nav1.2), paranode (MAG and connexin 32), juxtaparanode (Kv 1.2), and Schwann cell cytoplasm (S100b). Immunohistochemical analysis using antibodies against the selected proteins provided us with the anatomical arrangements of the two cell types in the nodes of Ranvier. We then examined the distribution of proteins involved in Ca2+ signaling within this architecture by using specific antibodies against IP3Rs, RyRs, P2Y1, M1, and Gq. In support of the original hypothesis, we found a highly concentrated distribution of Ca2+ signaling proteins in the paranodal loop regions on either side of the nodes of Ranvier (Toews et al., 2007). We plan to undertake a higher-resolution electron microscopic analysis to describe more precisely the colocalization of the two proteins in the axoglial apparatus. In future experiments, we plan to investigate, in collaboration with Mark Ellisman, the distribution of the signaling proteins at higher resolution by using electron microscopic immunocytochemical techniques. We will focus on co-localization of specific proteins in discrete regions of the Schwann cells.
Toews JC, Schram V, Weerth SH, Mignery GA, Russell JT. Signaling proteins in the axoglial apparatus of sciatic nerve nodes of Ranvier. Glia 2007;55;202-13.
In situ imaging of Schwann cell calcium signals
To measure directly the Schwann cell Ca2+ signals associated with action potential traffic along axons, we developed a transgenic mouse line that would express a fluorescent Ca2+ indicator photoprotein in a cell-specific manner in Schwann cells. We used the S100b promoter to target specifically a mutant chameleon protein, YC3.60, to Schwann cells in the peripheral nerves and astrocytes in the brain. We obtained a YC3.60 construct from Atsushi Miyawaki and engineered a plasmid construct containing the S100b promoter in tandem. YC3.60 is designed to express a protein containing the structures of Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), and calmodulin, a Ca2+ sensor. When the ambient Ca2+ concentration increases, the efficiency of fluorescent energy transfer (FRET) between CFP and YFP increases significantly, which is readily measured. Transfection of such a plasmid in C6 glioma and HEK 293 cell lines showed that YC3.60 was expressed only by C6 glioma cells, which express S100b, and not by HEK293 cells.
In collaboration with James Pickel, we used this plasmid to generate transgenic mouse founders. We established four founder lines expressing YC3.60 fluorescence in glial cells and bred them to homozygosity. We used two-photon confocal microscopy to image brain slice preparations obtained from two of the lines and readily visualized individual astrocytes brightly fluorescent with YC3.60 chameleon. We observed cells with large spongiform process arbors as well as smaller cells containing YC3.60. Many of the cells had elongated cell somas with several large branches radiating parallel or perpendicular to the branches of their neighboring glia. Numerous YC3.60-positive astrocytic processes extended to wrap around small blood vessels, and YC3.60 was expressed in the cell soma and processes in astrocytes. Dual staining with anti–S100b and anti–GFP antibodies in the cerebellum showed that about 78 percent of S100b-positive Bergmann glial cells expressed YC3.60.
To verify that the YC3.60 within cells functioned as a Ca2+ indicator in situ, we used acute brain slice preparations and previously characterized experimental paradigms to elicit neurotransmitter-induced glial cell Ca2+ signals. Several laboratories have demonstrated astrocytic Ca2+ signals in response to exogenously applied glutamate. Application of a puff of glutamate to astrocytes in a slice under superfusion elicited an increase in the YFP/CFP fluorescence ratio in the majority of the cells examined. In three different slice preparations obtained from heterozygous S100-YC-C mice, over half of the chameleon-expressing astrocytes in the microscopic field responded to the glutamate puff. The average glutamate-induced YFP/CFP ratio change was about 60 percent. In most experiments, one or two cells showed much larger YFP/CFP ratio changes (over 100 percent) than did the rest of the cells, whereas some cells did not respond at all. The largest observed response was a 300 percent change in the YFP/CFP ratio in a field where 12 of 13 cells examined showed an average response of about 100 percent. In another set of experiments, we examined Bergmann glial cells in cerebellar slices obtained from heterozygous S100-YC-C mice. Application of a puff of glutamate over Bergmann glial cells elicited an increase in YFP/CFP fluorescence ratios, indicating that cellular Ca2+ rises with a peak amplitude of about 57 percent, which was significantly lower than that recorded in astrocytes in cortical or hippocampal slices.
Stimulation of Schaffer collaterals in hippocampal slices evoked robust Ca2+ signals in astrocytes in the stratum radiatum and stratum moleculare-lacunosum. Schaffer collateral stimulation caused an immediate Ca2+ rise in a number of astrocytes in slice preparation. In slice preparations from S100-YC-C and S100-YC-H mice, the stimulus evoked a YFP/CFP ratio increase in about 77 percent of 74 cells, and, in each case, we recorded extracellular field potentials. The largest observed fluorescence increase was a 222 percent change in the YFP/CFP ratio, and the average response was about 60 percent. Using high-magnification objectives and electronic zoom, we then focused on one of the spongiform protoplasmic astrocytes. We repeated the stimulus paradigm during two-photon imaging and measured YC3.60 fluorescence changes within the indicated regions of interest. In the cell soma, a large increase in the YFP/CFP ratio followed the stimulus. Many of the regions of interest are glial microdomains within the amorphous spongiform morphology of the cell. The cellular Ca2+ response elicited by the neural stimulation spreads as a wave through the cell. Such glial microdomains of high activity are reminiscent of previously described small (less than 2µm) astrocytic terminal sheaths that enwrap single synapses or groups of synapses. It is likely that the discrete domains of high Ca2+ signals that we observed in our experiments represent spontaneous synaptic activity that excites terminal astrocytic branches ensheathing synapses.
One of the transgenic mouse lines showed abundant YC3.60 fluorescence within all Schwann cells in the peripheral nerves, whereas the astrocytes in the CNS of the mouse line did not show appreciable fluorescence. We propose to use mice derived from this line to investigate action potential–dependent Ca2+ signals in Schwann cells. Using infrared two-photon confocal microscopy during action potential propagation, we will image sciatic nerves isolated from the mice. We plan to hold isolated nerves by two suction electrodes, one to stimulate the nerve and the other to record compound action potentials. Multiphoton confocal microscopy allows for imaging deep within highly scattering tissue and permits the imaging of fluorescence changes in individual Schwann cells. Our experiments are expected to provide an answer to the question of whether action potential propagation in axons evokes specific cellular signals in Schwann cells. Following the initial characterization in isolated nerve preparations, we propose to image nerves in situ in the living mouse and record spontaneous intrinsic activity in axons. We will image Schwann cells by using two-photon confocal microscopy, record Schwann cell Ca2+ signals, and correlate the signals with electrical impulse conduction along axons.
Odling K, Albertsson C, Russell JT, Martensson LG. An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva. J Exp Biol 2006;209:956-64.
Ong HL, Liu X, Tsaneva-Atanasova K, Singh BB, Bandyopadhyay BC, Swaim WD, Russell JT, Hegde RS, Sherman A, Ambudkar IS. Relocalization of STIM1 for activation of store-operated Ca2+ entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca2+ store. J Biol Chem 2007;282:12176-85.
Weerth SH, Holtzclaw LA, Russell JT. Signaling proteins in raft-like microdomains are essential for Ca2+ wave propagation in glial cells. Cell Calcium 2007;41:155-67.
1 Stan Atkin, BS, former Student Training Fellow
COLLABORATOR
Mark Ellisman, PhD, National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA
Atsushi Miyawaki, MD, PhD, RIKEN, Tokyo, Japan
James Pickel, PhD, Laboratory of Genetics, NIMH, Bethesda, MD
For further information, contact james@helix.nih.gov.
